Tissue Clearing for 3D Cell Culture Models – ScaleS4, Visikol® HISTO-M™ and ClearT2

When researchers think of tissue clearing they generally think of the CLARITY technique and the beautiful spinning 3D mouse brain that Karl Deisseroth’s group at Stanford generated. However, tissue clearing for whole tissues such as whole mouse brains has remained largely an academic pursuit focused on very small sample sizes (i.e. one brain) for basis research studies and tissue clearing method papers.

Instead, one of the areas that tissue clearing has come to the forefront in is the characterization of 3D cell culture models (e.g. organoids, microtissues, spheroids) for use in drug discovery by pharmaceutical companies. These models provide improved in vivo relevancy compared to their 2D counterparts but are challenging to characterize due to their thickness and inherent opacity. Many researchers have attempted to characterize these models using traditional imaging approaches (e.g. confocal, widefield) which have been traditionally used for 2D adherent cell culture. Researchers that have attempted to characterize these models without tissue clearing tend to bias their results by only characterizing cells on the periphery of these models where cells are most proliferative and exposed to compound/nutrients. The reason for this is that the opacity of 3D cell culture models limits the penetration of light to 1-3 cell layers and thus internal cells cannot be characterized even if they are effectively labeled.

Therefore, researchers have begun to employ tissue clearing such that the entirety of these 3D cell culture models can be characterized. The context of 3D cell culture tissue clearing is very different than the context for the CLARITY technique developed by the Deisseroth group as 3D cell culture assays employ thousands of models per campaign which must be processed within wells on well plates. Conversely, the CLARITY technique was designed for a single brain at a time and to achieve high-resolution imaging of a very large tissue volume.

Therefore, techniques like CLARTY which require expensive equipment and a complicated protocol are not amenable for use with 3D cell culture models and are instead best suited for ultra-low-throughput/ultra-high-data-density basic research studies.. While solvent based techniques like i/3/u/vDISCO and BABB are rapid and compatible with immunolabeling, they are harsh solvents and will thus melt through the polystyrene well plates used with 3D cell culture models.

Aqueous techniques like ScaleS4 and CUBIC which employ urea to denature proteins have been shown to be amenable to high-throughput processing and are compatible with well plates. Recently, researchers from the the National Center for Advancing Translational Sciences (NCATS) demonstrated the use of ScaleS4 for nuclear morphology phenotypic screening. Through their work, they demonstrated a significant improvement in their ability to characterize the entirety of 3D cell culture models through the use of tissue clearing. However, one of the limitations with these urea based methods is that they render tissues transparent through the process of protein denaturation and are thus not compatible with immunolabeling without post-fixation and increase tissue fragility which can lead to disrupted models during pipetting.

To address these problems, we have developed Visikol HISTO-M which is a plate compatible solvent based tissue clearing technique that is compatible with immunolabeling as well as fluorescent protein and other chemical dyes. Additionally, Visikol HISTO-M is reversible which means that that for validation models can be imaged in 3D followed by traditional 2D histological sectioning and imaging.

Michael Johnson